Methyl-CoM reductase (MCR) catalyzes the final key step in methanogenesis. As a result, the determination of the MCR molecular mechanism is critical to the understanding of the unique metabolic pathway of methanogenesis. The objective of this proposal is to determine the catalytic role of the MCR cofactor, Ni F43O, during MCR catalysis. The specific aims to achieve this are the following: (a) to generate a fully active and stable form of MCR; (b) to characterize the catalytically competent Ni redox states of F430 and the interaction of methyl-SCoM with F430 during MCR catalysis; (c) to determine the overall catalytic mechanism of MCR and any rate limiting steps. The stopped-flow kinetic studies will include rapid freeze-quench techniques along with a variety of spectroscopic methods such a Uv- visible, EPR, and resonance Raman. The insights gained from the completion of these goals will contribute significantly to the understanding of the NCR molecular mechanism. It should be noted that I have recently isolated an active form of NCR which will allow me to begin to characterize the catalytic role of F430 while continuing to investigate the activation of MCR.